Enrichment of antigen-reactive T cells according to CD154 expression

Aim:
Ex vivo enrichment of CD154+ expressing T cells following stimulation with different antigens
in combination with intracellular cytokine staining

Materials:

RPMI-1640 medium
human AB-Serum
Brefeldin A
CD40 functional grade pur
CD28 functional grade pur
anti human CD154-Biotin
anti-Biotin Microbeads (CD154 Microbead Kit contains both components)
PEB-Puffer (1xPBS, 2 mM EDTA, 0,5% BSA) (keep buffer cold (4-8°C)
MACS MS Columns
antigens
Inside Fix solution
Inside Perm solution
1) In vitro stimulation for induction of CD154+ expression

• sterile preparation of PBMCs from buffy coats or whole blood via Ficoll
(if possible take fresh blood; we get the buffy coats in the evening, than I make PBMCs, put
them overnight in the incubator and stimulate the cells in the next morning)

• resuspend cells at a density of 1x107 cells/ml in culture medium (RPMI+5%AB serum)

• plate cells in dishes at a density of 5x106 cells/cm2

(usually we use 107 PBMCs in one well of a 24-Well plate; for very rare we start with 108
PBMCs in 2 wells of a 6-Well plate)

• add antigen in the appropriate concentration; include non-stimulated control

• add 1µg/ml CD40 blocking antibody (1:100)

• add 1 µg/ml CD28 functional grade antibody (1:100)

(if you expect really few antigen-specific cells, it is better not to use CD28 for a better signal-
to-noise ratio)

• incubate cells for 5 hours at 37°C, 5%CO2; add 1µg/ml BrefeldinA and incubate for
another 2hours
(it is important to keep this stimulation times to have enough time for CD154 to be up-
regulated!)

2) Magnetic labeling

• collect cells by pipetting up and down; rinse dish with cold buffer

• centrifuge at 300xg for 10min; aspirate supernatant

• labeling for 108 total cells: (for 107 total cells scale down volumes accordingly; keep titer
1:10 for biotin and bead labeling)

- 450µl PEB buffer + 50µl CD154-Biotin 10 min ; 4°C

- wash in 20 ml PEB buffer 10 min, 300xg
- 450µl PEB buffer + 50µl aBiotin-MB 15 min, 4°C
- wash in 20 ml PEB buffer 10 min, 300xg
- resuspend up to 108 cells in 500µl PEB buffer

3) Magnetic separation in combination with intracellular staining

- prepare MS columns by rinsing with 500µl PEB buffer

- apply cell suspension on the column
- wash column 2x with 500µl PEB buffer
- prepare staining mix for surface staining (total volume 70µl per column)
(it is important to have a total volume of 70µl staining solution; fill up to 70µl with PBS
buffer; it is good to include a dump channel for CD8/CD14/CD20/Live-Dead)
- apply surface staining Mix on the column and incubate 15 min at RT
- wash columns with 500µl PEB buffer
- elute cells with 500µl PEB buffer into 1.5ml Eppi
- add 250µl Inside Fix to the eluted cell fraction and incubate 15 min at RT

- prepare a second MS column by rinsing with 500µl PEB buffer

- apply fixed cell suspension directly on the second column
- wash columns with 500µl PEB buffer
- wash columns with 200µl Inside Perm buffer (Saponin)
- prepare staining mix for intracellular staining (total volume 70µl per column)
(it is important to have a total volume of 70µl staining solution; fill up to 70µl with Inside
Perm; CD154 can be stained intracellular)
- apply intracellular staining Mix on the column and incubate 10 min at RT
- wash columns with 200µl Inside Perm buffer
- elute cells with 1000µl PEB buffer into 1.5ml Eppi
- centrifuge cells for 5min at 300xg and take up in 50-100µl PEB buffer
- FACS analysis